PROJECT 2: Host-virus networks regulating flu replication and host responses ex vivo. Sumit Chanda, Project Leader; Megan Shaw, Co-Investigator; Ivan Marazzi, Co-Investigator, Nevan Krogan , Co-Investigator. In this proposal, we hypothesize that multiple, discrete molecular pathways determine influenza disease severity, and that these pathways elicit biomarker signatures that can be identified through network-based modeling of system-level measurements. In Project 2, we propose to utilize leading edge OMICS approaches to identify ex vivo molecular signatures that correlate with clinical disease outcomes. The elucidation and characterization of factors that govern outcome-related biomarker signatures offer the potential for the development of novel antiviral therapies. In Aim 1, we will use a systems biology approach to elucidate network signatures associated with clinical influenza severity. Here, we will profile changes to the host transcriptome, proteome and metabolome, in response to infection using viruses of different pathogenicity, as well as additional host perturbations linked to clinical outcome. Ex vivo molecular signatures correlated with disease severity will be integrated and modeled with in vivo and clinical data generated in Project 1. In Aim 2, we propose to use genetic tools to identify nodes within outcome-related molecular signatures that are critical regulators of host response pathways and viral replication (`driver genes'). Both Aims 1 and 2 will rely on a paradigm of reiterative experimentation and modeling to link ex vivo signatures to clinical phenotypes, and identify the host proteins and pathways that govern them. In Aim 3, those genes found to regulate pathways and signatures associated with disease outcome (driver genes) will be further characterized. We propose to employ CRISPR-based analysis of transcriptional, epigenetic, proteomic, and metabolic profiles to provide insight into the role of these factors in regulating responses linked to disease outcomes (CRISPR-OMICs). Additional molecular, cellular, biochemical and in vivo studies will be conducted to further characterize those nodes that determine disease outcomes as potential therapeutic targets.